Anti-angiogenic therapy has immense clinical potential to not only impede tumor growth but also to inhibit tumor metastases. However, a key challenge today in the development of new anti-angiogenic pharmaceuticals is the identification and validation of novel drug targets. Fortunately, our mode of action studies with the anti-angiogenic small molecule, TNP-470, identified a previously pharmaceutically-unexplored signaling pathway, i.e., the non-canonical Wnt Planar Cell Polarity (PCP) pathway, that we have demonstrated is required for angiogenesis. Using a combination of chemical genetic, murine knockout and zebrafish angiogenesis assays, we showed that inhibition of methionine aminopeptidase 2 (MetAP-2), the target of the fumagillin derivative TNP-470, results in loss of cellular polarity selectively in endothelial cells. Moreover, TNP-470- mediated inhibition of polarity blocks endothelial cell migration and proliferation (via a p53-dependent cell cycle arrest), ultimately leading to loss of angiogenesis. These results strongly suggest that inhibition of PCP signaling may be a viable pharmaceutical strategy for the development of new anti-angiogenic drugs. In this study, we plan 1) to characterize the molecular mechanisms by which MetAP-2 inhibition leads to loss of PCP signaling, 2) to explore the anti-angiogenic potential of other small molecule PCP inhibitors, and 3) to explore the potential clinical limitations of a PCP inhibition strategy, i.e., what undesired non-angiogenic consequences result from in vivo PCP inhibition. The combination of biochemical, small molecule, and murine model approaches proposed here will contribute new information regarding angiogenic regulation and may provide new targets for the development of additional anti-angiogenic agents. PUBLIC HEALTH RELEVANCE: Cancer continues to a major health concern in the U.S.. Since the number of new drugs approved each year continues to decline, it is important that we continue to pursue new lines of inquiry that could lead to novel anti-tumor pharmaceuticals. Our research explores new avenues of research that if successful would address this need for new anticancer drugs.